Malignant tumors and metastatic lesions of epithelial tissues are the most common form of cancer and are responsible for the majority of cancer-related deaths (Chambers et al., 2002; Pantel and Brakenhoff, 2004). Because of progress in the surgical treatment of these tumors, mortality is linked increasingly to early metastasis and recurrence, which is often occult at the time of primary diagnosis (Racila et al., 1998; Pantel et al., 1999). For example, the remote anatomical location of the pancreas and other gastrointestinal (GI) organs makes it unlikely that pancreatic and other GI cancers will be detected before they have invaded neighboring structures and grown to tumors larger than 1-cm (Compton, 2003; Flatmark et al., 2002; Koch et al., 2001; Liefers et al., 1998; Matsunami et al., 2003; Nomoto et al., 1998; Pantel et al., 1999; Walsh and Terdiman, 2003; Weihrauch, 2002). Even with respect to breast cancers, 12-37% of small tumors of breast cancer (<1 cm) detected by mammography already have metastasized at diagnosis (Chadha M et al., 1994; Wilhelm M C et al., 1991).
During the process of metastasis, cancerous cells detach from the primary tumor, migrate through the circulation, seed into secondary organs and grow a metastatic lesion. A rational strategy to diagnose cancer, therefore, would be to detect circulating tumor cells (CTC) in the peripheral blood after greater than 1,000-fold enrichment of metastatic tumor cells (Pantel et al., 1999). However, CTCs are currently detected by epithelial lineage markers or tumor-associated markers that are neither sufficient in distinguishing cancer from normal cells nor adequate in identifying specific types of tumors. For example, current CTC enrichment methods using an anti-Epithelial Cell Adhesion Molecule (EpCAM) antibody may entail a substantial loss of metastatic tumor cells due to downregulation of the EpCAM gene in these cells (Pantel et al., 1999; Sabile et al., 1999; Thurm et al., 2003; Choesmel et al., 2004). Therefore, gene expression profiles (GEP) of CTCs enriched by anti-EpCAM antibody magnetic cell separation may be misleading in that the cells in which they appear may be a selected subpopulation of CTCs that have upregulated EpCAM expression (Smirnov et al., 2005).
Molecular profiling of gene expression in primary and metastatic tumors is a powerful tool that can be used to diagnose tumor types and categorize them with respect to projected prognosis and therapeutic responsiveness (Berchuck et al., 2005; Jazaeri et al., 2005; Goto et al., 2006; Newton et al., 2006). The profiling is sometimes performed on tumor cells concentrated using antibodies against epithelial surface antigens in immuno-magnetic cell separation methods (Smirnov et al., 2005; Bayani et al., 2002; Tsuda et al., 2004; Yao et al., 2006; Shipitsin et al., 2007). In the past five years there have been more than 1,000 publications describing gene expression profiles of epithelial cancers mostly focusing on primary and secondary solid tumors. Only one paper compared gene expression profiles of cells isolated from blood of healthy subjects and cancer patients (Smirnov et al., 2005). The isolation step employed an antibody against the epithelial cell adhesion molecule (EpCAM). Antibodies against EpCAM (also known as tumor-associated calcium signal transducer 1, TACSTD1 or GA733) are commonly used in immuno-magnetic cell separation of CTCs, but EpCAM is expressed on both normal and malignant cells (Litvinov et al., 1994; Balzar et al., 1999; Winter et al., 2003). It is therefore not clear that the comparison made by Smirnov et al. was entirely valid.
Evidence has accumulated in the literature showing that epithelial tumor cells found in the circulation represent the earliest sign of metastasis formation and that circulating tumor cells (“CTCs”) can be considered an independent diagnostic for cancer progression of carcinomas (Beitsch and Clifford, 2000; Brandt et al., 2001; Feezor et al., 2002; Fehm et al., 2002; Ghossein et al., 1999; Glaves, 1983; Karczewski et al., 1994; Koch et al., 2001; Liefers et al., 1998; Luzzi et al., 1998; Matsunami et al., 2003; Molnar et al., 2001; Wang et al., 2000; Weitz et al., 1999; Wharton et al., 1999; Racila et al., 1998; Pantel et al., 1999). Accordingly, reliable procedures to isolate cancer cells from the bloodstream would have significant impact in both clinical diagnostic and therapeutic applications of cancer (Racila et al., 1998; Pantel et al., 1999). A new tumor staging, called Stage Mi, has been proposed to indicate the presence of tumor cells in the circulation of patients with cancers. The staging warrants the development of a blood test that could detect CTCs. However, CTCs are rare. In general, therefore, such tests require tumor cell enrichment methods that can increase detection sensitivity, advantageously by at least one order of magnitude (Pantel et al., 1999), over existing methods.
Cytokeratins (KRTs) are cytoskeletal proteins specifically expressed in cells of the epithelial lineage. They have become the standard markers for the detection of CTCs or tumor cells disseminated in bone marrow (DTCs) in patients with epithelial tumors such as breast, prostate, colon or lung cancer (Pantel et al., 2008; Paterlini-Brechot and Benali, 2007). Some CTCs are now being viewed as a type of stem or progenitor cell of epithelial lineage that differentiates to form secondary tumors (Braun et al., 2007; Shipitsin et al., 2007; Pantel et al., 2008; Paterlini-Brechot and Benali, 2007). However, it is not known to date which member(s) of the KRT family are specific for the cancer stem or progenitor cell type among CTCs. Indeed, some KRTs used in detecting CTCs were found to be expressed also in subpopulations of normal circulating cells (Pantel et al., 2008; Paterlini-Brechot and Benali, 2007). CTCs of epithelial cancers are thought to express markers of multiple cell lineages, including but not limited to stem cell genes or factors (SCFs), and are thought to be capable of proliferating and differentiating into the epithelial phenotype wherein not only cytokeratins (KRTs) are expressed but also cadherins (CDHs), mucins (MUCs), integrins (ITGs), epithelial membrane antigens (EMA) and tumor-associated antigens (TAAs). Although some of these differentiated epithelial markers have been used to isolate and identify CTCs and DTCs (Pantel et al., 2008), the true identity of the tumor progenitor cell population remains undefined for the majority of human cancers due to the lack of markers that are specific for that type of CTC (van't Veer and Bernards, 2008; Sawyers, 2008).
Current nucleic acid-testing by DNA microarray or quantitative real-time PCR (qPCR) has improved resolution in the detection of CTCs by means of cut-off values of “selected” marker transcript numbers, above which these transcripts can be considered as tumor cell-derived (Ignatiadis et al., 2008; Xenidis et al., 2006). There remains an unresolved quantification problem, however: there are no ‘ideal’ endogenous control genes; that is, genes that do not deviate between tumor and normal cells in blood from different individuals (de Kok et al., 2005).
Organ-specific markers (mammoglobulin, HER-2, CA-125, CEA and PSA) have been used to identify CTCs that have emigrated from their original organs such as breast, ovary, colon and prostate. However, false negative results can occur, since these antigens are not present in all the cells of a tumor (Paterlini-Brechot and Benali, 2007). In addition, these markers were not specific for a given organ. Gene expression analysis of a subpopulation of CTCs isolated by antibody against EpCAM from patients with metastatic cancer revealed that the KRT19 and AGR2 (hAG-2) genes were expressed in the majority of the metastatic samples, whereas S100A14, S100A16, and CEACAM5 genes showed expression restricted to the metastatic colorectal and breast cancer samples; FABP1 and KRT20 genes showed expression patterns associated with colorectal cancer; those of the SCGB2A1, SCGB2A2, and PIP genes were associated with breast cancer (Smirnov et al., 2005). Since EpCAM is commonly expressed on normal and malignant cells (Litvinov et al., 1994; Balzar et al., 1999; Winter et al., 2003) and EpCAM expression varies in different tumors (Braun et al., 1999; Thurm et al., 2003), the gene expression profiles of EpCAM-expressing circulating cells previously described (Smirnov et al., 2005) could target a subpopulation of circulating cells in metastatic cancer patients including normal inflammatory and lymphoid cells.
In cancer patients, the number of CTCs or exfoliated abnormal cells (neoplastic cells) in blood is generally very small compared to the number of non-neoplastic cells. Therefore, the detection of exfoliated abnormal cells by routine cytopathology is often limited. Further, exfoliated cells are frequently highly heterogeneous, being composed of many different cell types. Compounding this heterogeneity problem, the frequency of neoplastic cells present in each clinical specimen is variable, which biases and complicates the quantification of differential gene expression in randomized mixed populations. Apoptotic and necrotic cells are common in larger tumors, peripheral blood and ascites. These cells do not contain high quality RNA and thus present technical problems for molecular analyses (Karczewski et al., 1994).
The detection of metastatic cells is particularly challenging. Although primary cancers frequently shed neoplastic cells into the circulation at an early stage of metastases formation (Fidler, I. J., European Journal of Cancer 9:223-227, 1973; Liotta L. A. et al., Cancer Research 34:997-1004, 1974) in large numbers, only a minor subpopulation of such cells (one in a thousand to one in a million) are metastatic (Glaves, D., Br. J. Cancer 48:665-673, 1983), evidently because few shed cancer cells survive for long in the circulation (Weiss and Glaves, 1983; Karczewski et al., 1994)
A number of cell enrichment methods for circulating tumor cells have been described:                a) Microdissection can be used to isolate rare tumor cells one by one (Suarez-Quian et al., 1999). This method typically has several limitations: (1) the subsequent sample processing is complicated, (2) cell viability cannot readily be established, and (3) selection of the cells to be dissected is based mainly on morphological criteria, which has a high frequency of giving rise to false-positive results.        b) Physical characteristics of tumor cells, such as shape, size, density or electrical charge, can also be used (Vona et al., 2000). Several density gradient centrifugation methods have been developed to enrich tumor cells in nucleated blood cells (devoid of mature red blood cells). Density gradient centrifugation methods can achieve 500 to 1,000-fold cell enrichment. The enriched tumor cells can then be subjected to molecular analysis using highly sensitive assays such as immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) which may be used to amplify putative tumor markers or epithelial markers such as prostate specific antigen (PSA) mRNA or cytokeratin 19 mRNA (Peck et al., 1998). However, these methods may not effectively enrich viable tumor cells from normal cells. That is, 500-1,000 fold cell enrichment is often found to be relatively modest enrichment which generates substantial background noise adversely affecting further molecular analysis. In addition, enrichment methods based on physical separation techniques are often cumbersome, lengthy, and involve steps (e.g. more than 2-3 rounds of centrifugation) that can result in cellular damage.        c) Antibody-based techniques are a more recent development. Immunoaffinity methods include affixing an antibody to a physical carrier or fluorescent label. Sorting steps can then be used to positively or negatively enrich for the desired cell type after the antibody binds to its target present on the surface of the cells of interest. Such methods include affinity chromatography, particle magnetic separation, centrifugation, or filtration, and flow cytometry (including fluorescence activated cell sorting; FACS).        
Flow cytometry or a fluorescence activated cell sorter (“FACS”) detects and separates individual cells one-by-one from background cells. In model experiments, this method can detect breast carcinoma cells (Gross et al., 1995) and endothelial progenitor cells (Hill et al., 2003) in the mononuclear cell fraction that had been enriched from the peripheral blood by density gradient centrifugation. Furthermore, FACS can detect naturally occurring breast and prostate tumor cells in blood after an enrichment step using antibody-coated magnetic microbeads (Racila et al., 1998; Beitsch and Clifford, 2000). However, cells that exist in clusters or clumps are discarded during the FACS process, and in some instances, for example, ovarian cancer, most of the cells are present as aggregates, making FACS CTC or CEC detection highly ineffective.
Approaches based on antibody-coated microbeads can use magnetic fields (Racila et al, 1998), column chromatography, centrifugation, filtration or FACS to achieve separation. Despite its great power for enrichment, there are also inherent limitations associated with all of the antibody-based cell separation methods. The most serious one is that cancer cells usually express putative tumor-specific antigens to variable degrees (Sabile et al., 1999); hence it is easy to lose a large and potentially non-random subset of tumor cells during the collection. Antibodies also tend to bind with significant non-specific affinity to damaged cells, leading to their co-purification with the cells of interest. Overall, such antibody-based cell separation methods have a higher than desired false-negative rate. Current antibody-initiated magnetic separation methods have detected CTC at much lower levels, i.e., 1-100 CTC per mL of blood from patients with breast and prostate cancer (Racila et al., 1998). There are approximately 5×109 red cells and 5×106 white nucleate cells present in one milliliter (ml) or gram of blood. Therefore, it is still a challenging task to detect the presence of thousands of cancer cells in one ml of blood (Gulati and Acaba, 1993).
Over the past 20 years, specialized complexes found on the surface of invasive tumor cells that facilitate their movement from the primary tumor to sites of metastasis have been characterized (Aoyama and Chen, 1990; Chen and Chen, 1987; Chen et al., 1994a; Chen et al., 1984; Chen et al., 1994b; Chen, 1996; Chen, 1989; Chen and Wang, 1999; Ghersi et al., 2002; Goldstein and Chen, 2000; Goldstein et al., 1997; Kelly et al., 1994; Monsky et at, 1994; Monsky et al., 1993; Mueller et al., 1999; Mueller and Chen, 1991; Mueller et al., 1992; Nakahara et al., 1996; Nakaliara et al., 1998; Nakahara et al., 1997; Pavlaki et al., 2002; Pineiro-Sanchez et al., 1997; Saga et al, 1988; Zucker et al, 2000; Zukowska-Grojec et al., 1998). These complexes, which we have denoted as “invadopodia”, bind to and degrade multiple types of endothelial cell matrix (ECM) components. Invadopodia are not found on differentiated normal blood cells or on primary tumor cells, and they do not function effectively on dead or dying cells.
The applicant has recognized that an enrichment step based on invadopodia function would powerfully serve to separate viable metastatic tumor cells from the majority of cell types found in ascites, blood, and many other body fluids and would address the limitations of the other technologies described above. Apparatus and methods for conducting such an enrichment have been disclosed in U.S. Patent Application Publications 2002/0164825 published Nov. 7, 2002, 2003/0206901 published Nov. 6, 2003, 2005/0153342 published Jul. 14, 2005, 2005/0272103 published Dec. 8, 2005, all to Chen, and 2005/0244843 published Nov. 3, 2005, to Chen et al. All of the foregoing, and patents that issue therefrom, are incorporated herein in their entirety, by reference, for all purposes. The method employs a cell adhesion matrix (“CAM”) especially adapted to retain cells having invadopodia. Since the method can achieve a one million fold enrichment of such cell types over their concentrations in whole blood, all manner of molecular biological measurements can be made on them that would be impossible absent the enrichment step.
It is to be noted that invadopodia are present on circulating endothelial progenitor cells (but absent in more than 99.999% of blood cells), and in fetal cells found in maternal blood of pregnant females. Circulating endothelial cells (CECs) are progenitors for the cells covering the internal surface of blood and lymphatic vessels that are involved in clinical complications including cancer, cardiovascular diseases, autoimmune diseases, and infectious diseases (Tarin, 2006; Beerepoot et al., 2004; Hill et al., 2003). CECs tend to be enriched along with CTCs in the methods described in the references cited above.
Thus, cell samples so enriched comprise various types of CTCs (based on tumor of origin and on relative pluripotency), and CECs as well. Recently, gene expression profiling (GEP) of immuno-magnetically isolated CECs based on an antibody against CD146 (Smirnov et al., 2006) showed a distinct set of CEC genes that were different from those of immuno-magnetically isolated CTCs described in a previous paper (Smirnov et al., 2005). However, it is not possible to resolve these cell types in a mixed population due to a lack of markers specific for these cell types, be they cancer cells or endothelial cell progenitors (van't Veer and Bernards, 2008; Sawyers, 2008). Enrichment makes any particular cell type highly accessible to genetic analysis, but leaves one uncertain about the cell to which the analysis should apply. What is needed is a means of resolving these cell types.